Translocation Of HMGB1 Research Articles

4-cholesten-3-one suppresses lung adenocarcinoma metastasis by regulating translocation of HMGB1, HIF1α and Caveolin-1.

Metastasis is a great challenge in lung adenocarcinoma (ADC) therapy. Cholesterol has been implicated in ADC metastasis. 4-cholesten-3-one, as cholesterol metabolite and analog, can substitute membrane cholesterol and increase membrane fluidity. In this study, we explored the possibility that 4-cholesten-3-one inhibited ADC metastasis. Low-dose 4-cholesten-3-one significantly restrained ADC cells migration and invasion with little effects on cells viabilities. Further investigation showed that 4-cholesten-3-one promoted ROS generation, which transiently activated AMPKα1, increased HIF1α expression, reduced Bcl-2 expression and caused autophagy. AMPKα1 knockdown partly suppressed 4-cholesten-3-one-induced autophagy but, neither prevented 4-cholesten-3-one-induced upregulation of HIF1α or downregulation of Bcl-2. 4-cholesten-3-one-induced autophagy facilitated the release of HMGB1 from nuclei to cytoplasm, blocking nuclear translocation of HIF1α and activation of MMP2 and MMP9. Also, 4-cholesten-3-one induced time-dependent phosphorylation of caveolin-1, Akt and NF-κB. With increasing treatment time, 4-cholesten-3-one accelerated caveolin-1 internalization, but reduced the phosphorylation of Akt and NF-κB, and inhibited the expression of snail and twist. These data suggested that 4-cholesten-3-one could be a potential candidate for anti-metastasis of lung adenocarcinoma.

Nle4, D-Phe7]-α-MSH Inhibits Toll-Like Receptor (TLR)2- and TLR4-Induced Microglial Activation and Promotes a M2-Like Phenotype.

α-melanocyte stimulating hormone (α-MSH) is an anti-inflammatory peptide, proved to be beneficial in many neuroinflammatory disorders acting through melanocortin receptor 4 (MC4R). We previously determined that rat microglial cells express MC4R and that NDP-MSH, an analog of α-MSH, induces PPAR-γ expression and IL-10 release in these cells. Given the great importance of modulation of glial activation in neuroinflammatory disorders, we tested the ability of NDP-MSH to shape microglial phenotype and to modulate Toll-like receptor (TLR)-mediated inflammatory responses. Primary rat cultured microglia were stimulated with NDP-MSH followed by the TLR2 agonist Pam3CSK4 or the TLR4 agonist LPS. NDP-MSH alone induced expression of the M2a/M2c marker Ag1 and reduced expression of the M2b marker Il-4rα and of the LPS receptor Tlr4. Nuclear translocation of NF-κB subunits p65 and c-Rel was induced by LPS and these effects were partially prevented by NDP-MSH. NDP-MSH reduced LPS- and Pam3CSK4-induced TNF-α release but did not affect TLR-induced IL-10 release. Also, NDP-MSH inhibited TLR2-induced HMGB1 translocation from nucleus to cytoplasm and TLR2-induced phagocytic activity. Our data show that NDP-MSH inhibits TLR2- and TLR4-mediated proinflammatory mechanisms and promotes microglial M2-like polarization, supporting melanocortins as useful tools for shaping microglial activation towards an alternative immunomodulatory phenotype.

Progranulin protects against endotoxin-induced acute kidney injury by downregulating renal cell death and inflammatory responses in mice

Progranulin (PGRN), a pluripotent secreted growth factor, is involved in various physiologic and disease processes. However, the role of PGRN in endotoxin-induced septic acute kidney injury (AKI) remains unknown. The objective of this study is to investigate the protective effects of PGRN on an endotoxin-induced AKI mouse model by using PGRN-deficient mice and recombinant PGRN (rPGRN) pretreatment. PGRN levels were increased in kidneys of wild-type (WT) mice at 6 and 24h after lipopolysaccharide (LPS) injection. Renal function detection, hematoxylin and eosin staining, immunohistochemical staining, ELISA and in situ terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick-end labeling were used to reveal tissue injury, inflammatory cell infiltration, production of inflammatory mediators and cell death in mouse kidneys after LPS injection. PGRN deficiency resulted in severe kidney injury and increased apoptotic death, inflammatory cell infiltration, production of pro-inflammatory mediators and the expression and nucleus-to-cytoplasmic translocation of HMGB1 in the kidney. In addition, rPGRN administration before LPS treatment ameliorated the endotoxin-induced AKI in WT mice. PGRN may be a novel biologic agent with therapeutic potential for endotoxin-induced septic AKI possibly by inhibiting LPS-induced renal cell death and inflammatory responses in mice.

Decreased neuroinflammation correlates to higher vagus nerve activity fluctuations in near-term ovine fetuses: a case for the afferent cholinergic anti-inflammatory pathway?

BackgroundNeuroinflammation in utero may contribute to brain injury resulting in life-long neurological disabilities. The pivotal role of the efferent cholinergic anti-inflammatory pathway (CAP) in controlling inflammation, e.g., by inhibiting the HMGB1 release, via the macrophages’ α7 nicotinic acetylcholine receptor (α7nAChR) has been described in adults, but its importance in the fetus is unknown. Moreover, it is unknown whether CAP may also exert anti-inflammatory effects on the brain via the anatomically predominant afferent component of the vagus nerve.MethodsWe measured microglial activation in the ovine fetal brain near term 24 h after the umbilical cord occlusions mimicking human labor versus controls (no occlusions) by quantifying HMGB1 nucleus-to-cytosol translocation in the Iba1+ and α7nAChR+ microglia. Based on multiple clinical studies in adults and our own work in fetal autonomic nervous system, we gauged the degree of CAP activity in vivo using heart rate variability measure RMSSD that reflects fluctuations in vagus nerve activity.ResultsRMSSD correlated to corresponding plasma IL-1β levels at R = 0.57 (p = 0.02, n = 17) and to white matter microglia cell counts at R = −0.89 (p = 0.03). The insult increased the HMGB1 translocation in α7nAChR+ microglia in a brain region-dependent manner (p < 0.001). In parallel, RMSSD at 1 h post insult correlated with cytosolic HMGB1 of thalamic microglia (R = −0.94, p = 0.005), and RMSSD at pH nadir correlated with microglial α7nAChR in the white matter (R = 0.83, p = 0.04). Overall, higher RMSSD values correlated with lower HMGB1 translocation and higher α7nAChR intensity per area in a brain region-specific manner.ConclusionsAfferent fetal CAP may translate increased vagal cholinergic signaling into suppression of cerebral inflammation in response to near-term hypoxic acidemia as might occur during labor. Our findings suggest a new control mechanism of fetal neuroinflammation via the vagus nerve, providing novel possibilities for its non-invasive monitoring in utero and for targeted treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0567-x) contains supplementary material, which is available to authorized users.

Adiponectin Inhibits LPS‐Induced HMGB1 Release through Heme Oxygenase‐1 Dependent Pathway in RAW 264 Cells

IntroductionHigh mobility group protein B1 (HMGB1) is an important late inflammatory mediator in sepsis. Adiponectin, an adipokine predominantly secreted from adipose tissue, has potent anti‐inflammatory properties. However, the possible anti‐inflammatory effects of adiponectin on lipopolysaccharides (LPS)‐induced HMGB1 release are unknown. The aim of this study was to investigate whether adiponectin could inhibit HMGB1 release in LPS‐stimulated RAW 264 cells.Materials and methodsMurine macrophage RAW 264 cells were treated with or without LPS (200 ng/ml) in the presence or absence of adiponectin, interleukin (IL)‐10, HO‐1 inhibitor (ZnPP) and p38 MAPK inhibitor (SB203580). HMGB1 translocation and release were measured by immunofluorescence staining of the cells and western blotting analysis of the medium, respectively. Expression levels of mRNA for toll like receptor (TLR) 4, TLR2, myeloid differentiation factor‐2 (MD‐2), HMGB1, IL‐10, heme oxygenase‐1(HO‐1) and nuclear factor erythoid‐derived 2 related factor 2 (Nrf2) mRNA were assessed by real‐time PCR.Results and discussionTreatment of the cells with LPS induced significant HMGB1 release and obvious translocation from nucleus to cytosol. However, prior treatment with either adiponectin or IL‐10 suppressed the LPS‐induced HMGB1 release and translocation. Adiponectin treatment decreased TLR4 mRNA and induced expression of HO‐1 mRNA, but not IL‐10 mRNA, while IL‐10 treatment decreased TLR2 and HMGB1 mRNA and enhanced both IL‐10 and HO‐1 gene expressions. Treatment of RAW 264 cells with ZnPP before adiponectin and IL‐10 treatment obviated the suppression of HMGB1 release by adiponectin completely, but partially by IL‐10 while treatment with SB203580 didn't had a significant effect. In conclusion, our results indicate that adiponectin suppresses HMGB1 release by LPS through a HO‐1‐dependent while neither HO‐1 nor p38 MAPK was involved in IL‐10 anti‐inflammatory effect. Therefore, both adiponectin and IL‐10 may play a role as an anti‐inflammatory molecule in the late inflammatory process by preventing release of a septic exaggeration factor, HMGB1.

Inhibition of the translocation and extracellular release of high-mobility group box 1 alleviates liver damage in fibrotic mice in response to D-galactosamine/lipopolysaccharide challenge.

Acute liver injury in the setting of fibrosis is an area of interest in investigations, and remains to be fully elucidated. Previous studies have suggested the beneficial effects of liver fibrosis induced by thioacetamide and partial bile duct ligation against Fas-mediated acute liver injury. The activation of AKT and extracellular signal-regulated kinase signaling is considered to be crucial in this hepatoprotection. To demonstrate the protection of CCl4-induced liver fibrosis against lethal challenge, the present study compared the reactivity to lethal doses of D-galactosamine (D-GalN)/lipopolysaccharide (LPS) between fibrotic mice and control mice groups. The extent of hepatic damage was assessed by survival rate and histopathological analysis. The molecular basis of the fibrosis-based hepatoprotection was examined, with a particular focus on the translocation and release of high-mobility group box (HMGB)1 and the inflammatory response triggered by HMGB1. Hepatoprotection induced by fibrosis was demonstrated by improved survival rates (100%, vs. 20%) and improved preservation of liver architecture in fibrotic mice subjected to D-GalN/LPS, compared with control mice treated in the same way. D-GalN/LPS evoked the translocation and release of HMGB1, detected by immunohistochemistry, in the control mice, which was significantly inhibited in the fibrotic mice. The gene expression levels of HMGB1-associated proinflammatory cytokines, including interleukin (IL)-1β, IL-6, tumor necrosis factor-α and IL-12p40, were markedly inhibited in the fibrotic mice when exposed to D-GalN/LPS. These findings confirmed that CCl4-based fibrosis induced hepatoprotection, and provided evidence that fibrosis inhibited the translocation and release of HMGB1, and the proinflammatory response triggered by HMGB1. This alleviated liver damage following exposure to D-GalN/LPS challenge.

Dioscin attenuates renal ischemia/reperfusion injury by inhibiting the TLR4/MyD88 signaling pathway via up-regulation of HSP70

We previously reported the effect of dioscin against hepatic ischemia/reperfusion injury (IRI) in rats. However, little is known concerning the role of dioscin in renal IRI. In the present study, rats were subjected to IRI and dioscin was intragastrically administered for seven consecutive days before surgery. In vitro models of hypoxia/reoxygenation were developed in NRK-52E and HK-2 cells, which were prophylacti -cally treated with or without dioscin. The results showed that dioscin significantly decreased serum BUN and Cr levels, and markedly attenuated cell injury. Mechanistic studies showed that dioscin significantly increased HSP70 levels, decreased the levels of TLR4, MyD88, TRAF6, COX-2, JNK, ERK and p38 MAPK phosphorylation, suppressed the nuclear translocation of NF-κB and HMGB1, and subsequently decreased the mRNA levels of IL-1β, IL-6, TNF-α, ICAM-1 and IFN-γ. Moreover, HSP70 siRNA or TLR4 DNA reversed the nephroprotective effects of dioscin, while dioscin still significantly down-regulated the TLR4 signaling pathway. Furthermore, by inhibiting MyD88 with ST2825 (a MyD88 inhibitor), renal IRI was significantly attenuated, suggesting that the effect of dioscin against renal IRI depended on MyD88. Our results suggested that dioscin had a potent effect against renal IRI through suppressing the TLR4/MyD88 signaling pathway by up-regulating HSP70. These data provide new insights for investigating the natural product with the nephroprotective effect against IRI, which should be developed as a new therapeutic agent for the treatment of acute kidney injury in the future.

HMGB1 and RAGE in skeletal muscle inflammation: Implications for protein accumulation in inclusion body myositis

Inflammation is associated with protein accumulation in IBM, but precise mechanisms are elusive. The “alarmin” HMGB1 is upregulated in muscle inflammation. Its receptor RAGE is crucial for β-amyloid-associated neurodegeneration. Relevant signaling via HMGB1/RAGE is expected in IBM pathology. By real-time-PCR, mRNA-expression levels of HMGB1 and RAGE were upregulated in muscle biopsies of patients with IBM and PM, but not in muscular dystrophy or non-myopathic controls. By immunohistochemistry, both molecules displayed the highest signal in IBM, where they distinctly co-localized to intra-fiber accumulations of β-amyloid and neurofilament/tau. In these fibers, identification of phosphorylated Erk suggested that relevant downstream activation is present upon HMGB1 signaling via RAGE. Protein expressions of HMGB1, RAGE, Erk and phosphorylated Erk were confirmed by Western blot. In a well established cell-culture model for pro-inflammatory cell-stress, exposure of human muscle-cells to IL-1β+IFN-γ induced cytoplasmic translocation of HMGB1 and subsequent release as evidenced by ELISA. Upregulation of RAGE on the cell surface was demonstrated by immunocytochemistry and flow-cytometry. Recombinant HMGB1 was equally potent as IL-1β+IFN-γ in causing amyloid-accumulation and cell-death, and both were abrogated by the HMGB1-blocker BoxA. The findings strengthen the concept of unique interactions between degenerative and inflammatory mechanisms and suggest that HMGB1/RAGE signaling is a critical pathway in IBM pathology.

Xenon Treatment Protects against Remote Lung Injury after Kidney Transplantation in Rats.

Ischemia-reperfusion injury (IRI) of renal grafts may cause remote organ injury including lungs. The authors aimed to evaluate the protective effect of xenon exposure against remote lung injury due to renal graft IRI in a rat renal transplantation model. For in vitro studies, human lung epithelial cell A549 was challenged with H2O2, tumor necrosis factor-α, or conditioned medium from human kidney proximal tubular cells (HK-2) after hypothermia-hypoxia insults. For in vivo studies, the Lewis renal graft was stored in 4°C Soltran preserving solution for 24 h and transplanted into the Lewis recipient, and the lungs were harvested 24 h after grafting. Cultured lung cells or the recipient after engraftment was exposed to 70% Xe or N2. Phospho (p)-mammalian target of rapamycin (mTOR), hypoxia-inducible factor-1α (HIF-1α), Bcl-2, high-mobility group protein-1 (HMGB-1), TLR-4, and nuclear factor κB (NF-κB) expression, lung inflammation, and cell injuries were assessed. Recipients receiving ischemic renal grafts developed pulmonary injury. Xenon treatment enhanced HIF-1α, which attenuated HMGB-1 translocation and NF-κB activation in A549 cells with oxidative and inflammatory stress. Xenon treatment enhanced p-mTOR, HIF-1α, and Bcl-2 expression and, in turn, promoted cell proliferation in the lung. Upon grafting, HMGB-1 translocation from lung epithelial nuclei was reduced; the TLR-4/NF-κB pathway was suppressed by xenon treatment; and subsequent tissue injury score (nitrogen vs. xenon: 26 ± 1.8 vs. 10.7 ± 2.6; n = 6) was significantly reduced. Xenon treatment confers protection against distant lung injury triggered by renal graft IRI, which is likely through the activation of mTOR-HIF-1α pathway and suppression of the HMGB-1 translocation from nuclei to cytoplasm.

HMGB1 translocation is involved in the transformation of autophagy complexes and promotes chemoresistance in leukaemia.

Acute lymphoblastic leukaemia (ALL) is a common paediatric cancer and is among the most curable cancers. However, the acquisition of drug resistance is a significant obstacle to the achievement of favourable outcomes, and autophagy is regarded as a mechanism that underlies chemoresistance. In this study, RT-qPCR was used to measure the expression of HMGB1 and Beclin1 in bone marrow mononuclear cells. A CCK-8 test was conducted to assess cell viability. Western blot, immunofluorescence and transmission electron microscopic analyses were performed to evaluate the autophagy levels. Immunoprecipitation analysis was performed to detect protein-protein interactions in the autophagy complexes. We found that HMGB1 expression correlated with the clinical status of ALL. In vitro, anticancer agent-induced cytotoxic effects were associated with autophagy-related drug resistance, and these effects were ameliorated by FIP200 depletion or the application of autophagy inhibitors. Moreover, the Ulk1‑Atg13-FIP200 complex, which promotes HMGB1 trafficking, acted upstream of the HMGB1-Beclin1 and PI3KC3-Beclin1 complexes and played a critical role in autophagy. Targeting the transformation of autophagic complexes or HMGB1 translocation may suppress autophagy and consequently overcome chemoresistance in leukaemia.

434. Coxsackievirus A11 Induces Robust Oncolytic Activity Against Human Non-Small Cell Lung Cancer Cells With Immunostimulatory Properties

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Innovative treatment is therefore required to improve overall survival rates of advanced NSCLC. Oncolytic virotherapy using enteroviruses emerges as a promising anticancer strategy. As therapeutic advantages, enteroviruses immediately induce robust oncolytic activity and do not have oncogenes that may lead to tumorigenesis.Herein, we focused on enterovirus strain of coxsackievirus A11 (CVA11) to assess its oncolytic activity in a panel of human NSCLC cell lines because it has low pathogenicity but possesses broad oncolytic activity in our first screening assay using 38 enteroviruses. We found that CVA11 infection exhibited extensive oncolytic activity in 7 of 7 human NSCLC cell lines in a MOI-dependent manner, whereas it displayed no cytotoxicity in HaCaT normal human keratinocyte cell line. As CVA11 belongs to the species human enterovirus C that utilizes ICAM-1 for cell entry, we investigated the correlation of ICAM-1 expression levels and CVA11-mediated cytotoxicity, revealing a tendency that NSCLC cells expressing higher levels of ICAM-1 exhibited higher CVA11-mediated cytotoxicity. Gain-of-function assay showed that RD cells overexpressing human ICAM-1 acquired a susceptibility to CVA11 infection resulting in robust cellular destruction even at a low MOI of 0.01, whereas parental RD cells with negligible ICAM-1 showed no cytotoxicity, illustrating that ICAM-1 molecule could be one of cell surface receptors for CVA11 infection.To dissect the role of apoptotic cell death in CVA11-mediated oncolysis, we assessed the impact of pretreatment with Z-VAD, a pan-Caspase inhibitor which prevents apoptosis of the CVA11-driven cytotoxicity. Apoptosis partially but significantly was demonstrated to contribute to the CVA11-driven oncolysis in NSCLC cell lines. Furthermore, our results of xenograft models using nude mice transplanted with human NSCLC tumors revealed that intratumoral CVA11 administrations significantly suppressed the outgrowth of preestablished xenografts and prolonged the survival of the CVA11-treated mice. Of note, all mice treated with CVA11 showed no significant changes of body weight during the treatment. Finally we investigated whether CVA11 infection could induce immunogenic cell death by releasing DAMPs such as calreticulin (CRT) exposure and HMGB1 translocation. Of note, CVA11 infection induced abundant surface exposure of CRT in a time dependent manner and robust release of HMGB1 from the nuclei of NSCLC cells into the cytosol.We showed that CVA11 displayed remarkable oncolytic activity against human NSCLC with tolerability and immunostimulatory properties indicating a promising oncolytic therapeutics against NSCLC. Further preclinical and clinical studies are required for the translation of this new modality to clinical use.

Sodium arsenic and ultraviolet-radiation synergistically induce cell injury in lens and retinal epithelial cells occurring migration of HMGB1 (IRM11P.639)

Abstract Although sodium arsenic (As) and ultraviolet radiation (UVR) can induce cell injury through either DNA damage or reactive oxidative species (ROS) in various epithelial cells, whether damaged-associated molecular pattern (DAMP) involved in cell injury is still unclear. To determine the association of HMGB1, one of DAMP molecules that can be secreted by oxidative stress, we investigated cell injury in human lens epithelial cell (HLE B3) and human retinal epithelial cell (ARPE-19) using cell viability assay, western blot, confocal microscopy and flow cytometry (FACS analysis). In both HLE B3 and ARPE-19 cells, DNA damage marker, cleaved PARP and cell viability assay showed that UVR synergistically increased As-caused cell death(injury). During UVR and As-caused cell injury, HMGB1 was translocated from nucleus to cytosol and then secreted to extracellular region. ROS scavenger N-acetylcystein (NAC) and mito-tempo inhibited both translocation and secretion of HMGB1 in dose-dependent manner, implicating that migration of HMGB1 is closely associated with ROS generation in mitochondria. Taken together, we concluded that HMGB1 is associated in cell injury caused by UVR and As. Nevertheless how migrated HMGB1 plays a role in UVR-As caused cell injury of HLE B3 and arpe-19 cells is still unclear.

Respiratory syncytial virus infection triggers HMGB1 release from lung epithelial cells to promote inflammatory cytokine production (CAM1P.152)

Abstract Respiratory syncytial virus (RSV) is an important cause of lower respiratory tract infections in infants and young children. Oxidative stress is shown to play an important role in the pathogenesis of lung inflammation. We have shown that RSV induces oxidative stress by rapid generation of reactive oxygen species and downregulation of antioxidant enzyme expression resulting in lung inflammation. High mobility group box 1 protein (HMGB1) is a redox-sensitive multifunction protein that serves as both a chromatin protein and an extracellular signaling molecule. Although HMGB1 has been implicated in inflammatory diseases, there has been no report on its role in RSV related lung disease. The goal of this study is to dissect the mechanisms that induce the release of HMGB1 and associated cellular signaling in response to RSV infection. Human lung epithelial cells were infected with RSV and harvested over time to analyze HMGB1 expression by Western blot, immunohistochemistry and qRT-PCR. Our studies show for the first time that RSV infection induces the translocation of HMGB1 from cell nuclei to the cytoplasm and its subsequent release to the extracellular space demonstrating a novel mechanism of HMGB1 in the activation of cells to promote inflammatory cytokine release. Understanding of HMGB1 role in the setting of viral-induced oxidative stress may lead to the development of novel strategies to treat respiratory viral infections in pediatric and elderly population.

Effects of propofol on lipopolysaccharide-induced expression and release of HMGB1 in macrophages.

This study aimed to determine the effects of different concentrations of propofol (2,6-diisopropylphenol) on lipopolysaccharide (LPS)-induced expression and release of high-mobility group box 1 protein (HMGB1) in mouse macrophages. Mouse macrophage cell line RAW264.7 cells were randomly divided into 5 treatment groups. Expression levels of HMGB1 mRNA were detected using RT-PCR, and cell culture supernatant HMGB1 protein levels were detected using enzyme-linked immunosorbent assay (ELISA). Translocation of HMGB1 from the nucleus to the cytoplasm in macrophages was observed by Western blotting and activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus was detected using ELISA. HMGB1 mRNA expression levels increased significantly in the cell culture supernatant and in cells after 24 h of stimulating RAW264.7 cells with LPS (500 ng/mL). However, HMGB1 mRNA expression levels in the P2 and P3 groups, which received 500 ng/mL LPS with 25 or 50 μmol/mL propofol, respectively, were significantly lower than those in the group receiving LPS stimulation (P<0.05). After stimulation by LPS, HMGB1 protein levels were reduced significantly in the nucleus but were increased in the cytoplasm (P<0.05). Simultaneously, the activity of NF-κB was enhanced significantly (P<0.05). After propofol intervention, HMGB1 translocation from the nucleus to the cytoplasm and NF-κB activity were inhibited significantly (each P<0.05). Thus, propofol can inhibit the LPS-induced expression and release of HMGB1 by inhibiting HMGB1 translocation and NF-κB activity in RAW264.7 cells, suggesting propofol may be protective in patients with sepsis.

Dioscin ameliorates cerebral ischemia/reperfusion injury through the downregulation of TLR4 signaling via HMGB-1 inhibition

We previously reported the promising effect of dioscin against hepatic ischemia/reperfusion (I/R) injury, but its effect on cerebral I/R injury remains unknown. In this work, an in vitro oxygen–glucose deprivation and reoxygenation (OGD/R) model and an in vivo middle cerebral artery occlusion (MCAO) model were used. The results indicated that dioscin clearly protected PC12 cells and primary cortical neurons against OGD/R insult and significantly prevented cerebral I/R injury. Further research demonstrated that dioscin-induced neuroprotection was accompanied by a significant inhibition in the expression and the nuclear to cytosolic translocation of HMGB-1, reflected by decreased TLR4 expression. Blockade of the TLR4/MyD88/TRAF6 signaling pathway by dioscin inhibited NF-κB and AP-1 transcriptional activities, MAPK and STAT3 phosphorylation, and pro-inflammatory cytokine responses, and upregulated the levels of anti-inflammatory factors. In addition, small interfering RNA (siRNA) and overexpressed genes of HMGB-1 and TLR4 were applied in in vitro experiments, respectively, and the results further confirmed that dioscin showed an efficient neuroprotection because of its inhibiting effects on HMGB-1/TLR4 signaling and subsequent suppressing inflammation. These findings provide new insights that will aid in elucidating the effect of dioscin against cerebral I/R injury and support the development of dioscin as a potential treatment for ischemic stroke.

High-mobility group box-1 was released actively and involved in LPS induced depressive-like behavior

Depression disorder is a common mental illness, of which the pathogenesis is not well understood. Studies suggest that immunity imbalance and up-regulation of pro-inflammatory cytokines may be associated with the pathogenesis of depression. High-mobility group box 1 protein (HMGB1) has gained much attention as an important player in innate immune responses and an modulating factor in several inflammatory diseases. Here we sought to explore the role of HMGB1 in the development of depression. Depression model was established with low dose of lipopolysaccharide (LPS) administration. Depressive behavior was reflected with increased immobility time in tail suspension test. Accompanying with depressive-like behavior, translocation of HMGB1 from nuclei to cytoplasm was observed by immunofluorescence assays. Meanwhile, no significant necrosis was observed evaluated by hematoxylin-eosin staining. These data indicated that HMGB1 was released actively in the central nervous system. In addition, treating the mice with human recombinant HMGB1 (rHMGB1) could induce the development of depressive-like behavior. Blockage of HMGB1 with GZA abrogated the depressive-like behavior induced by LPS or rHMGB1. These results implicated that HMGB1 was involved in LPS-induced depressive-like behavior.

Abstract 202: Involvement of neuronal high mobility group box 1 (HMGB1) in Angiotensin II-mediated neuronal-microglial interaction

We have demonstrated that activation of microglia in the autonomic brain regions, primarily in the paraventricular nucleus (PVN), is critical in the development and establishment of hypertension. Neuroimmune synaptic communication is implicated in activating microglia and inducing neuroinflammation in many neuropathophysiological diseases. Specifically, HMGB1 secreted by neurons is a critical mediator in neuroimmune crosstalk. Thus we hypothesized that HMGB1 is a key regulator involved in microglia activation in Ang II-induced hypertension. Neuronal and microglial cells in primary culture were established for this study. Effect of Ang II on microglial movement was recorded and quantified by time-lapse microscopy and video analysis. In addition, the expression, translocation and secretion of HMGB1 in neurons were measured by qPCR, Western blot and immunocytochemistry. Ang II treatment of neuronal-microglial co-culture resulted in a 5-fold increase of microglial migration towards neurons, which was completely abolished by treatment with 1 μM Losartan, an Ang II receptor type 1 antagonist. The presence of neurons was found to be essential for microglial movement since Ang II failed to induce these effects in the absence of neurons. Neurons express high levels of HMGB1, which was predominantly localized in the nucleus. Ang II treatment resulted in a time-dependent translocation of nuclear hMGB1 into the cytoplasm and ultimately into the medium. Both translocation and release of Ang II-induced HMGB1 was blocked by Losartan. Furthermore treatment of microglia with 500ng/ml recombinant HMGB1 caused ~2 fold increase of proinflammatory cytokines (TNF-α and IL-1β). The importance of HMGB1 is further underscored by our observation that its protein level was increased ~2 fold in the PVN but not in the nucleus of the solitary tract (NTS) or rostral ventrolateral medulla (RVLM) of the spontaneously hypertensive rats (SHR) compared with the normotensive WKY rats. Taken together, these observations suggest that Ang II primarily activates the neuronal AT1R to increase the expression, cytosolic translocation and release of HMGB1. The released HMGB1 primes microglial activation and release of cytokines, two key events in neuroinflammation in hypertension.

A novel androstenedione derivative induces ROS-mediated autophagy and attenuates drug resistance in osteosarcoma by inhibiting macrophage migration inhibitory factor (MIF).

Osteosarcoma is a common primary bone tumor in children and adolescents. The drug resistance of osteosarcoma leads to high lethality. Macrophage migration inhibitory factor (MIF) is an inflammation-related cytokine implicated in the chemoresistance of breast cancer. In this study, we isolated a novel androstenedione derivative identified as 3,4-dihydroxy-9,10-secoandrosta-1,3,5,7-tetraene-9,17-dione (DSTD). DSTD could inhibit MIF expression in MG-63 and U2OS cells. The inhibition of MIF by DSTD promoted autophagy by inducing Bcl-2 downregulation and the translocation of HMGB1. N-acetyl-L-cysteine (NAC) and 3-methyladenine (3-MA) attenuated DSTD-induced autophagy but promoted cell death, suggesting that DSTD induced ROS-mediated autophagy to rescue cell death. However, in the presence of chemotherapy drugs, DSTD enhanced the chemosensitivity by decreasing the HMGB1 level. Our data suggest MIF inhibition as a therapeutic strategy for overcoming drug resistance in osteosarcoma.

The expression of HMGB1 on microparticles from Jurkat and HL-60 cells undergoing apoptosis in vitro.

HMGB1 is a highly conserved nuclear protein that displays important biological activities inside as well as outside the cell and serves as a prototypic alarmin to activate innate immunity. The translocation of HMGB1 from inside to outside the cell occurs with cell activation as well as cell death, including apoptosis. Apoptosis is also a setting for the release of cellular microparticles (MPs), which are small membrane-bound vesicles that represent an important source of extracellular nuclear molecules. To investigate whether HMGB1 released from cells during apoptosis is also present on MPs, we determined the presence of HMGB1 on particles released from Jurkat and HL-60 cells induced to undergo apoptosis in vitro by treatment with either etoposide or staurosporine; MPs released from cells undergoing necrosis by freeze-thaw were also characterized. As shown by both Western blot analysis and flow cytometry, MPs from apoptotic cells contain HMGB1, with binding by antibodies indicating an accessible location in the particle structure. These results indicate that HMGB1, like other nuclear molecules, can translocate into MPs during apoptosis and demonstrate another biochemical form of this molecule that may be immunologically active.

Genistein Increase Intracellular Distribution of the High Motility Group Box-1 through p38 Pathway in HeLa culture cells induced by Tumor Necrosis Factor-α

Cervical cancer is one kind of many cancer that cause death to women around the world. Many studies had support the statement that inflammation has a strong linkage with cancer development. Several factors like proinflammatory factor can influence tumor cell microenvironment, and induce a faster proliferation. TNF-&alpha; is suspected can induce proliferation. While cancer itself can induce inflammation, which is marked by several marker. One of them is HMGB1, released from the cell as active secretory lysosomes or passive diffusion. Genistein has demonstrated growth inhibitory effects of various types of cancer cells. It inhibits tyrosine kinase pathway, which can be activated by TNF-&alpha;. One of those pathways that have the link with proliferation is p38. This study tries to reveal about inhibitory effect of genistein toward p38 pathway that had been activated by TNF-&alpha;. This research was conducted by exposing cultured HeLa cells with various doses of genistein for 90 minutes, and then exposed to TNF - &alpha; 10 ng / mL for 20 minutes. Observations were made with a confocal microscope, by staining the cells with pp38-TRITC and HMGB1 antibody. The intensity was measured and analyzed by Fluoview software. The results suggest that there be significant differences between pp38 intranuclear intensity and HMGB1 extranuclear intensity of each dose of genistein (p = 0.000, ANOVA). pp38 and HMGB1 intensity were increased along with increasing genistein dose, but at high dose there were noted decreasing of pp38 and HMGB1 intensity. At apoptotic dose, pp38 and HMGB1 intensity were increased markedly, showing the effect of apoptosis. In general, increasing doses of genistein increase intranuclear p38 activation and HMGB1 extranuclear translocation. So there were a strong linkage between p38 activation and HMGB1 translocation in this study.